Bone is a dynamic tissue that remodels itself continuously by both deposition of new bone and resorption of bone cells. The cells responsible for formation of bone tissue are known as osteoblasts, while the cells involved in bone resorption are known as osteoclasts. In some situations, however, resorption of bone is increased resulting in formation of abnormal bone structure. Diseases associated with an abnormal level of bone resorption include Paget""s disease, osteoporosis, and tumor-linked bone resorption disease.
Osteoclasts are located on the surface of mineralized bone. When bone is resorbed, calcium and phosphorus are released into the extracellular fluid and the organic matrix of bone is resorbed. In Paget""s disease, bone resorption is usually focal. The osteoclasts in patients with Paget""s disease are triggered to undergo excessive resorption which is followed by replacement of normal bone marrow with vascular, fibrous connective tissue. As the disease progresses, the resorbed bone itself is replaced by coarse-fibered, dense trabecular bone that is organized haphazardly. This haphazard bone remodeling leads to a characteristic mosaic pattern in bone rather than the organized pattern seen in healthy bone tissue. The cause of this disease is unknown and there are currently no treatments that can reverse disease and few that effectively treat this disease. There are also no animal or cellular models currently available to study the etiology or treatment of Paget""s disease.
Although the exact etiology of Paget""s disease is unknown, the increased osteoclast activity is considered the hallmark of the disease (Roodman, G. D. et al., J. Clin. Invest., 1992, 89:46-52). Studies have shown that osteoclast number is abnormally high in active Paget""s disease lesions (Mills, B. G. et al., Bone, 1994, 15:443-448). In addition, bone marrow accessory cells isolated from pagetic bone lesions increased the number of CD34+ colony forming units, markers for hematopoietic cells which is the cell lineage of osteoclasts, in both normal and pagetic bone marrow (Demulder, A. et al., Endocrinology, 1993, 133:1978-1982). These data suggest that increased bone resorption in Paget""s disease is due to an increased number of osteoclasts as well as increased activity of these cells.
Information on the nature and activity of pagetic osteoclasts is limited by the difficulty of obtaining human osteoclasts from bone and by the small numbers of osteoclasts which can be obtained by in vitro bone culture methods (Demulder, A. et al., Endocrinology, 1993, 133:1978-1982). To date, no human cell line of osteoclasts has been established that would be useful in studying the activity of these cells in Paget""s disease and well as diseases such as osteoporosis or tumor-linked bone resorption disease.
Small numbers of osteoclasts have been produced in long term bone marrow culture (Kurihara, N., C. Civin, and G. D. Roodman, J., Bone Miner. Res., 1991, 6:257-261; Bayever, E. K. Haines, and S. F. Silverton, J. Bone Miner. Res., 1991, 6:S93). In addition, small numbers of osteoclasts have been observed in long term culture of spleen cells along with either osteoblast or bone-derived cell lines (Takahashi, N. N. Udagawa, T. Akatsu, H. Tanaka, M. Shionome, and T. Suda, J. Bone Miner. Res., 1991, 6:977-985; Takahashi, N., T. Akatsu, N. Udagawa, T. Sasaki, A. Yamaguichi, J. M. Moseley, T. J. Martin, and T. Suda, Endocrinology, 1988, 123:2600-2602). These multinucleate tartrate-resistant acid phosphatase-positive cells have several characteristics which resemble an isolated osteoclast, including the ability to form resorption pits in calcified tissue. However, these cell lines which appear to generate osteoclast cells required more than one cell type to produce the bone-resorbing cells (Takahashi, N., T. Akatsu, N. Udagawa, T. Sasaki, A. Yamaguichi, J. M. Moseley, T. J. Martin, and T. Suda, Endocrinology, 1988, 123:2600-2602; Burger, E. H., J. W. M. van der Meer, and P. J. Nijweide, J. Cell Biol., 1984, 99:1901-1906; Udagawa, N., N. Takahashi, T. Akatsu, T. Sasaki, A. Yamaguichi, H. ,Kodama, T. J. Martin, and T. Suda, Endocrinology, 1989, 125:1805-1813; Shinar, D. M., M. Sato, and G. A. Rodan, Endocrinology, 1990, 126:1728-1735). These cell line data suggested that cell to cell contact between an osteoclast precursor and the support cell was required for development of cells with osteoclast activity, i.e., bone resorptive capacity. None of this research, however, has demonstrated the production of a human cell line of osteoclast or osteoclast-like cells that is derived solely from one cell type.
When osteoclast-like cells are obtained by either isolation from human bone or in vitro cell culture, a functional characterization is required to differentiate osteoclasts from closely related macrophages or giant cells. Osteoclasts, but not other cells, are able to excavate focal areas of a calcified substrate when they are adherent to the calcified substrate (Jones, S. J. and A. Boyde, Bone, 1993, 14:455-460). Confirmation of this focal resorptive capacity requires analysis of calcified tissue destruction as evidenced by increase in pit number, pit area, and pit volume in the calcified substrate.
The present invention provides an osteoclast-like human cell line using bone cells from Paget""s patients.
An object of the present invention is to provide a human osteoclast-like cell line wherein the cell line is capable of resorbing calcified tissue in vitro.
Another object of the present invention is to provide a method for screening agents for treatment of bone resorption-related diseases comprising establishing a human osteoclast-like cell line which is adherent to a sample of calcified tissue, contacting the cell line with an agent to be screened, and determining whether the agent decreases resorption of the calcified tissue sample. Also claimed are methods for screening agents for treatment of the specific diseases associated with increased bone resorption that includes Paget""s disease, osteoporosis, and tumor-linked bone resorption disease.
Yet another object of the present invention is to provide an in vitro model for Paget""s disease of bone.
The present invention is a human xe2x80x9costeoclast-likexe2x80x9d cell line. xe2x80x9cOsteoclast-likexe2x80x9d cells are cells of a single type that were derived from pagetic bone tissue and have the ability to cause resorption of calcified tissue in vitro. These cells provide an in vitro model for Pagent""s disease.
Cells were transformed from pagetic bone using large xe2x80x9cTxe2x80x9d antigen and the immortalized cell line of the present invention was created. The cell line was shown to have the capacity to resorb calcified tissue, the unique characteristic of these cells, by culturing the cell lines on coverslips and dentin slices (the calcified tissue source). In addition to carrying out focal resorption of calcified tissue in vitro, these cell lines express colony stimulating factors such as M-CSF and G-CSF, and have the presence of other osteoclast markers including tartrate resistance acid phosphatase and carbonic anhydrase. In addition, these cell lines are able to support the development of osteoclasts from normal bone marrow in vitro.
Human pagetic bone tissue was removed during osteotomy to correct a significant bone defect caused by long-standing Paget""s disease of bone. The cells were prepared for culture and transfection. Cells were infected with a retrovirus containing the coding sequence for SV40 T antigen packaged in the PA317 cell line. The human pagetic cells were then grown and maintained and passaged continuously every 3 weeks. After 18 months, all cell lines increased their proliferative rate significantly and were then passaged once a week.
The characteristics of the cells were then determined. Expression of T antigen was assayed by immunofluorescence. Expression of CD34 antigen was assayed with a monoclonal antibody against human CD34 conjugated to fluorescein. The cell lines were positive for both antigens, consistent with osteoclast cells that had been transfected with T antigen.
Cell morphology was consistent with that of osteoclasts. Both pyramidal and round cells were seen in all cultures, at a ratio of about 20:1. All cells were grown on glass coverslips in the presence of a dentin (calcified tissue) disc. When adherent to the dentin discs, these pagetic cell lines were positive for osteoclast markers such as tartrate-resistant acid phosphatase, carbonic anhydrase, and succinic dehydrogenase. Some cells were positive for the vitronectin receptor. The cells lines were also shown to express M-CSF and G-CSF but not GM-CSF, IL-6 or TNF-alpha.
After all assays were performed for analysis of markers in the cell lines, the dentin slices were removed from culture and washed for resorption pit analysis. The pits were visualized with Toluidine Blue stain and the defects created by the cells were observed and counted using either light microscopy or scanning electron microscopy.
At a seeding density of 2xc3x97104, pagetic cell lines showed pit densities with a range of 2-68 and a median of 10 pits per 0.2 cm2 after 7-10 days. Pit areas were widely distributed. Pit depths, standardized by atomic force microscopy, ranged from 0.03 to 0.9 microns, with a median of 0.27 microns. Pit volumes ranged from 5 to 1141 cubic microns.
Pagetic cell lines were also co-cultured with adherent human bone marrow cells. Bone marrow cells had been collected after informed consent during bone marrow harvesting in normal subjects for bone marrow transplantation. These bone marrow cells were also plated in wells containing a dentin slice and cultured before addition of cells from the pagetic cell lines. After co-culture, the number of tartrate-resistant acid phosphatase cells was determined. The number of these marker cells increased with co-culture as compared to culture of bone marrow cells without pagetic cell lines added. This is the first demonstration of such an effect in a human osteoclast-like cell line. The immortalized cells demonstrated the capacity to increase the yield of bone resorptive cells from undifferentiated normal bone marrow. Such an effect is characteristic of pagetic cells and may explain in part the increased resorptive capacity that is typically seen in this disease.
The cell lines of the present invention are the first human osteoclast-like cell lines characterized and shown to have the ability to induce bone resorption in calcified tissue. The cell lines of the present invention are useful in the study of Paget""s disease and for screening agents that might be active to decrease the bone resorptive capacity of osteoclasts in this disease. The cell lines are also useful in the study of other diseases characterized by increased bone resorption, also termed bone resorption-related diseases, such as osteoporosis and tumor-linked bone resorption disease, or any condition where the activity of osteoclasts has been implicated.
Another embodiment of the present invention is a screening assay for agents to be tested for the treatment of a bone resorption-related disease. The diseases include Paget""s disease, osteoporosis, and tumor-linked bone resorption disease. In this screening assay, an osteoclast-like cell line of the present invention is established that is adherent to a sample of calcified tissue, for example a dentin slice. Then the cell line is contacted with an agent to be screened for treatment of a bone resorption-related disease. The endpoint to be determined in the assay is whether the test agent affects the resorption of calcified tissue in vitro, when compared to resorption in cell lines without addition of the test agent. Agents that would be useful in the treatment of such bone resorption-related diseases would decrease the level of bone resorption in the calcified tissue. Bone resorption can be measured by methods well known to those of skill in the art, including a pit assay using microscopic examination of pits on the calcified tissue sample. Levels of test agents to test in the screening assay would be chosen based on the knowledge of one of skill in the art of in vitro drug screening. Such doses typically range from 10xe2x88x929 M to 10xe2x88x924 M.
The following nonlimiting examples are provided to further illustrate the present invention.